This invention relates to magnetically-responsive particles, magnetically-responsive fluorescently-tagged particles, and the fabrication of such particles. The methods described can be used to fabricate particle populations that are distinguishable from each other by fluorescence and/or magnetic response. Multiple particle populations thus constructed will find utility in a number of fields, including clinical biological assays.
Microspheres have long been used as substrates on which to perform chemical reactions.
Catalysts for chemical modifications, such as hydrogenation or hydroformylation, can be attached to polymer beads to facilitate separation of the catalyst from the reaction products (U.S. Pat. No. 4,506,030, incorporated herein by reference). It is also a well known technique to couple biological molecules to the surface of a microsphere to assay the presence or absence of a reacting species in a biological sample; an example of such an assay would be an antigen-antibody reaction (U.S. Pat. No. 5,948,627, incorporated herein by reference).
This assay system can be improved by conducting the reaction on a microsphere (or other carrier particle) that has been labeled with a fluorescent material. The use of fluorescent labels on or in the microspheres allows preparation of numerous distinguishable sets of microspheres, based on different dye emission spectra or signal intensity. In the case of their use in a biological assay, these particles can then be analyzed on a flow cytometer to classify the size and fluorescence of the particles, as well as the fluorescence associated with the assay system being studied (e.g., a fluorescently labeled antibody in a xe2x80x9ccapture sandwichxe2x80x9d assay)(U.S. Pat. No. 5,948,627, incorporated herein by reference). This concept has been developed even further by incorporating multiple dyes in a particle, and creating distinguishable sets by using varying concentrations of these dyes. In this manner, hundreds, or even thousands, of different microsphere sets can be produced. In an assay, each different microsphere set would be associated with a different target, thus allowing numerous tests to be conducted on a single sample in a single container (U.S. Pat. No. 5,981,180, incorporated herein by reference).
Another method of modifying the particle is to incorporate a magnetically responsive substance, such as Fe3O4, into the structure. Such particles may be manufactured in a number of ways. For example, by adding the magnetic substance to the reaction vessel prior to initializing the polymerization that forms the particle (U.S. Pat. No. 4,339,337, incorporated herein by reference). In this case the magnetically responsive material is dispersed throughout the polymeric particle. A variation of this concept involves encapsulation of one or more magnetic particles in a hydrophobic polymeric shell (U.S. Pat. No. 5,356,713, incorporated herein by reference). Another method utilizes pH-induced precipitation of metal salts onto and into the pores of a polymeric microsphere (U.S. Pat. No. 4,774,265, incorporated herein by reference). The resulting particle may then be further coated with polymer to fix the magnetic material into the final particle. No attempt has been made to make particles with different magnetic response so that different populations would be discernable.
Magnetic particles such as these have found a number of uses in biomedical research and diagnostics. Antibodies targeted to specific cells can be coupled to magnetic microspheres, then, upon exposure to a biological sample, these cells can be selectively removed and collected by applying a magnetic field, towards which the target cell-microsphere pair will migrate (U.S. Pat. No. 4,230,685, incorporated herein by reference). Similarly, immunoassays may be performed on the surface of magnetic beads, and the magnetic field is applied to immobilize the microparticle during a wash step.
Magnetically-responsive fluorescent particles have also been made (EP 0463144B1). Magnetic particles may be directly dyed by either covalently coupling a dye to the surface of the particle, or by absorbing a hydrophobic dye into the particle. The former option has the disadvantage of the dye being exposed to the surrounding environment, and it is known that varying this solvent environment often results in spectral changes for the dye. When the fluorescent intensity must be tightly controlled, these spectral changes can not be tolerated. Absorption of the dye into the particle allows the dye to be exposed to a consistent environment, thus providing consistent spectral properties, but the dyeing process involves the use of organic solvents, which are often incompatible with the polymer used to form the particle. A polymer such as polystyrene can be used if it also contains a cross-linker, such as divinylbenzene. However, the layered construction of many magnetic particles results in a microsphere, that, upon immersion in organic solvents which leads to a swelling of the polymer, often loses its sphericity or some of its magnetic component. Depending on the desired use, this may or may not be acceptable.
Another approach is to add the magnetic material to a dyed particle. This can be accomplished through the salt precipitation technique described earlier, and, if the dye in the particle is not sensitive to the reaction conditions, results in an adequate particle. It may, however, be necessary to add another layer of polymeric material to the particle in order to immobilize the magnetic component. This is usually accomplished with a free radical initiated polymerization, which often causes dye decomposition.
This invention relates to particles, and methods of making particles, with a desired magnetic response. The term xe2x80x9cparticlexe2x80x9d refers to a core particle, for example a microsphere or bead, associated with at least one magnetic material. The core particle may also, for example, be associated with reactants, fluorescent tags, and other materials known in the art to be useful with core particles. The term xe2x80x9cmagnetic,xe2x80x9d as used hereinafter, includes all types of materials that respond to magnetic fields, such as, but not limited to, ferromagnetic, paramagnetic, and superparamagnetic materials. The term xe2x80x9cmagnetic materialxe2x80x9d encompasses any material having at least some magnetic content and therefore includes material having an amount of magnetic material ranging from greater than 0% to 100%. In some embodiments, the magnetic material is a polymeric magnetic material. Polymeric magnetic material includes for example, material in which the magnetic material is mixed with polymeric material and magnetic material which is coated with polymeric material. The term xe2x80x9creactantxe2x80x9d encompasses any substance capable of associating with at least one other substance, and which is used to identify or quantitate an analyte in solution. For example, antibodies and antigens can both be reactants. Surface reactive moieties such as amines, thiols, carboxylic acids, hydrazines, halides, alcohols, and aldehydes are also non-limiting examples of reactants. The term xe2x80x9canalytexe2x80x9d refers to any substance suspected of being present in a sample.
In one aspect, the present invention provides particles having a desired magnetic response. The phrase xe2x80x9cmagnetic responsexe2x80x9d refers to attractive or repulsive forces as a result of the application of a magnetic field. The magnetic response can be, for example, migration rate or retention time in response to a magnetic field. xe2x80x9cDesiredxe2x80x9d implies that the amount and type of magnetic material associated with the core particle are chosen by the skilled artisan to provide a core particle having a magnetic response suitable to achieve the skilled artisan""s preferred end result. The migration rate need not be quantitative (e.g. distance/time), but may be qualitative (e.g., faster or slower than another population of particles). So too, retention time may be quantitative or qualitative. The phrase xe2x80x9cpopulation of particlesxe2x80x9d refers to a set of particles that are similar, for example in magnetic response and other classification parameters (such as fluorescent intensity) where relevant, to the extent they can be identified as belonging to that population.
In one aspect, the present invention provides methods for controlling the amount of magnetic material associated with particles thereby controlling the magnetic response of the particles.
In one aspect, the present invention provides methods for forming populations of particles, each population having a different desired magnetic response. The magnetic response relates to the type and amount of magnetic material. In some embodiments, each population uses a different magnetic material, but the amount of magnetic material used per particle is about the same for each population. In some embodiments, each population uses the same magnetic material but varies the amount. For example, the magnetic material can be in the form of a nanosphere and each population uses the same size nanospheres, but a different number of nanospheres per particle, or else each population can use a different size nanosphere but the same number of nanospheres per particle, or else a combination of varying the size and number of nanospheres as long as each population has a different desired magnetic response.
In one aspect, the invention provides fluorescently-tagged particles having a desired magnetic response. In some embodiments, those xe2x80x9chybridxe2x80x9d particles have very defined fluorescent and magnetic responses. The phrase xe2x80x9cvery definedxe2x80x9d implies that there is little variation in the fluorescent and magnetic signal from bead to bead in a population. xe2x80x9cLittle variationxe2x80x9d means that the particles of one population can be distinguished from the particles of another population. In some embodiments xe2x80x9clittle variationxe2x80x9d means that the fluorescent intensities of the particles are within 10% of one another and the magnetic response of particles of one population can be distinguished from the magnetic response of particles of another population.
In one aspect, the present invention provides populations of particles and pooled populations of those particles, wherein one population is distinguishable from another population based at least on the magnetic response of the particles within a population.
In one aspect, the present invention provides methods for making particles with a desired magnetic response. In some embodiments, the desired magnetic response is achieved by associating magnetic material of chosen average size with core particles. In some embodiments, the desired magnetic response is achieved by associating a chosen amount of magnetic material with core particles. The chosen amount, or magnetic content, can be controlled by varying the magnetic content of the magnetic material associated with the core particles, and/or by varying the total amount of magnetic material associated with the core particles.
In one aspect, the present invention provides a method of making particles having a desired magnetic response by associating magnetic nanoparticles with the core particles. The term xe2x80x9cnanoparticlexe2x80x9d and xe2x80x9cnanospherexe2x80x9d are used interchangeably.
It will be apparent to one of ordinary skill in the art, from the description in this specification, that specific embodiments of the present invention may be directed to one, some or all of the above-indicated aspects as well as other aspects, and may encompass one, some or all of the above- and below-indicated embodiments as well as other embodiments. Thus, for example, a method according to the present invention may comprise making particles have a desired magnetic response by choosing both the size of the magnetic nanoparticles, the content of the nanoparticles, and the content of magnetic material associated with each core particle.